Mutations result from a variety of spontaneous processes and environmental insults. The determination of mechanisms holds the best promise for the understanding and possible treatment/prevention of genetic diseases. Recently, several groups have shown an effect of transcription on mutation. In the model organism, Saccharomyces cerevisiae, high levels of gene transcription lead to elevated mutation rates, a phenomenon termed Transcription-Associated Mutation (TAM). Specifically, elevated transcription through the LYS2 gene produces a unique mutational signature at LYS2: 2-nt deletions and, to a lesser degree, other mutation types. Moreover, at least some transcription-associated mutation types require the translesion synthesis polymerase, DNA polymerase zeta (Pol ?), for occurrence; thus, transcription-associated mutation likely occurs at sites of spontaneous DNA damage. The overall goal of this proposal is to characterize the mechanism } or mechanisms - of transcription-associated mutation and ultimately understand its role in genetic instability. New mutation assays designed to detect specific mutation types will permit quantitative studies to determine whether specific mutation yields are altered and also allow qualitative studies to determine whether the classes of mutations (i.e., the mutational spectra) and/or their distributions have been altered. Furthermore, the new assays will allow genetic approaches to identify roles of specific proteins or DNA lesions in the occurrences of specific classes of transcription-associated mutations. In Aim 1, new base substitution assays will be used to determine the effect of elevated transcription level on base substitution mutagenesis. Aim 2 proposes to determine the role of a major spontaneous DNA lesion, 8-oxo-7,8-dihydrodeoxyguanosine, in transcription-associated mutation. In Aim 3, a new assay will be used to determine the effect of elevated transcription level on gross chromosomal rearrangements. Transcription-associated mutation may play an important role in the generation of mutations in non- dividing cells. As such, it may contribute to a wide range of human diseases including cancer, aging, and neurodegenerative disorders. The work proposed here will clarify the breadth of TAM and begin to identify its underlying mechanism(s) - critical steps toward understanding its overall influence on human health. [unreadable] [unreadable] [unreadable]